Abstract This proposal uses a nanotechnology and optical imaging-based strategy for prevention (AIM 1) and image- guided treatment (Aim 2) of breast cancer (BrCa) by targeting several key molecular targets in 3D cultures and orthotopic murine models. In Aim 1, for BrCa prevention, we propose to use nanotechnology for enhancing outcome by improving bioavailability and limiting toxicity of chemo-preventive agents which include selective estrogen receptor antagonists, aromatase inhibitors, retinoids, COX-2 inhibitors and metformin. The strategy is to use Chemopreventive Nanocells (CPNC) that encapsulate raloxifene, anastrazole, metformin, fenretinide and celecoxib as preventive agents with indocyanine green (ICG) as the imaging agent. In Aim 2, from a treatment perspective the proposal exploits nanotechnology encapsulated chemotherapy for systemic cytotoxic therapy, photodynamic therapy (PDT) for localized cytotoxicity and simultaneous biological therapy to improve the treatment outcomes against HER2 positive and triple negative BrCa. The underlying hypothesis is that cytotoxic therapies combined with simultaneous intracellular targeting of multiple BrCa-associated molecular markers will be an effective combination therapy and will be most useful when incorporated with an imaging system that guides the treatment. The selected markers for the study range from secreted cytokines, vascular endothelial growth factor (VEGF), to membrane bound receptors, epidermal growth factor receptors (EGFR and HER2), their intracellular counterparts (intracellular VEGF ,EGFR and HER2), and the sub-nuclear protein PARP. Our surprising findings of dramatically enhanced tumor cell killing in vitro and in vivo when VEGF or EGFR is targeted intracellularly form the basis of the proposed study. Our strategy is to use a Theranostic Nanocell (TNC) that includes a PDT agent (benzoporphyrin derivative/ BPD, FDA approved agent) and biological agents (Avastin, Cetuximab, Herceptin, Lapatanib and Olapraib) as therapeutics with ICG as the diagnostic component. Nab-paclitaxel, a FDA approved nanoparticle formulation of paclitaxel forms the systemic therapeutic arm. We will use a hyperspectral fluorescence microscope capable of non-invasively imaging BPD and ICG for simultaneous, background-free, quantitative in vivo visualization of breast tumors. This will allow us to establish the pharmacokinetics of TNC and its biodistribution, disease assessment and the initiation of light treatments thus helping enhance overall survival. If successful, this study will impact women at high risk for developing breast cancer, primary BrCa patients or those with residual disease at the time of surgery, as well as women with recurrent disease. The technology development of CPNC and TNC is adaptable to other cancers with potential for impact on cancer prevention and treatment. Deliverables include i) CPNC and TNC fabrication and dosing; ii) 3D and in vivo orthotopic BrCa models for therapeutic screening. This provides the potential for rapid screening to establish individualized patient-specific molecular targets and forms the basis for designing personalized prevention and treatments.